A human tissue-based assay identifies a novel carrion blowfly strain for maggot debridement therapy

Maggot debridement therapy (MDT) is a form of therapeutic wound treatment in which live fly larvae are used intentionally to debride necrotic tissues. MDT has been widely used to treat chronic wounds in humans or animals, such as diabetic foot ulcers. Larvae of a carrion blowfly, Lucilia sericata (green bottle fly), debride wounds by consuming necrotic tissue and removing pathogenic bacteria, promoting effective wound healing. Most medical L. sericata strains were initially collected from natural environments using animal meat as bait and reared on artificial protein-rich media or ground meat. It remains to be examined which strain would be more appropriate for MDT, whereas any method for evaluating the fly’s therapeutic potential in humans has not been available. A feeding assay was developed using minced human tissues obtained from surgical waste. To establish L. sericata strains highly eligible for MDT, carrion fly larvae were collected from 45 corpses subjected to forensic autopsy (such as decomposed bodies). Four corpse-derived L. sericata strains were obtained and evaluated using the feeding assay. One strain showed that its feeding activity was 1.4 times higher than the control strain used in conventional MDT. The body length of the adult fly of the corpse-derived strain was longer than the control, which was consistent with the observation that its cell size was enlarged. The human tissue-based assay developed in this study accurately evaluated the ability of fly larvae to debride necrotic wounds. The L. sericata strain newly established from human corpses harboring high feeding activity may offer a clinically significant improvement in MDT.

Human tissue. Human tissues were collected as medical waste from approximately sixty free-flap surgeries. The surgeries occurred between August 2015 and May 2017 at The Jikei University Hospital, Tokyo, Japan. The Ethics Committee of The Jikei University School of Medicine determined that informed consent was not required because this study used human tissues obtained from medical waste, and personal information was firmly anonymized by mixing those tissues during the mincing process. Information about this study was published on a bulletin board at The Jikei University Hospital. Normal tissues were sorted according to the organ such as skin, muscle, fat, and bone and stored at − 80 °C. Frozen tissues were defrosted once, minced using an electric meat grinder SG-50 (Fukunou Sangyo Co., Ltd., Hyogo, Japan) three times, and stored at − 80 °C until the feeding assays. Necrotic tissues were also collected from three free flap reoperations. These necrotic tissues, including skin, muscle, and fat, were pooled, stored at − 80 °C, and minced as described above. Due to the condition of tissue collecting, the tissues used in this study were not wholly sterile, and certain bacteria are assumed to be present.
Porcine and bovine tissue. Pork-beef mince (a mixture of ground beef and pork meat) and pork fat mince were used in this study. Pork mince, beef mince, and pork fat tissue were purchased from Hanamasa Co., Ltd. (Tokyo, Japan). To prepare pork-beef mince, pork and beef minces were mixed at a one-to-one ratio and stored www.nature.com/scientificreports/ at − 20 °C. Pork fat tissue was minced using an electric meat grinder SG-50 (Fukunou Sangyo Co., Ltd., Hyogo, Japan) three times and stored at − 20 °C.
Fly strain. A L. sericata strain available in Japan for medical use was obtained from Japan Maggot Company Ltd. (Okayama, Japan) and used as a control strain in this study. In order to establish new fly strains for MDT, dipteran larvae were collected from human corpses, which were subject to forensic autopsy in the western Tokyo area (Tama), Japan, from 2015 to 2016 (Table 1). Each larva was fed with pork-beef mince, separated into a plastic tube after pupation, and grown to adulthood. Species identification of fly. Fly morphological traits were used for estimating fly species. Images of the whole body of each adult were taken using a stereomicroscope S6E (Leica Microsystems GmbH, Wetzlar, Germany) equipped with an ILCE-5100 camera (Sony Corporation Ltd., Tokyo, Japan) with a NY-1S adaptor (Micronet Inc., Saitama, Japan). Morphological characteristics (number of para-vertical setulae and occipital bristles, color of basicosta, and number of setae on the scutum of the mesonotum) were used to distinguish each fly image as reported previously 31,32 .
Fly species identified as L. sericata or L. cuprina by morphological traits were then confirmed by sequencing mitochondrial and nuclear genes as follows. The whole body of each adult fly was used for DNA purification after obtaining its offspring. DNA was extracted using a conventional method: each fly was homogenized in 200 µl of Buffer A (0.1 M Tris [pH 9.0], 0.1 M EDTA, 1% SDS, and 0.5% DEPC), and the homogenized mixture was incubated at 70 °C for 30 min. After adding 44.8 µl of 5 M potassium acetate, the mixture was incubated on ice for 30 min. The supernatant was collected by centrifugation at 20,400×g at 4 °C for 15 min and mixed with 90 µl of isopropanol. Precipitated DNA was collected after centrifugation at 20,400×g at 4 °C for 20 min, rinsed with 70% ethanol, and dried. DNA was diluted in TE and prepared for sequencing. Three genes were chosen for sequencing: mitochondrial cytochrome oxidase subunit I (COI), 28S rRNA (28S), and period (per). The primers 5′-CTG CTA CTT TAT GAG CTT TAG G-3′ and 5′-CAT TTC AAG YTG TGT AAG CAT C-3′ reported previously 33 were used to amplify a region of approximately 350 bp of the COI gene. The primer pairs, 5′-CCC CCT GAA TTT AAG CAT AT-3′ and 5′-GTT AGA CTC CTT GGT CCG TG-3′, and 5′-GCC TTC AGA TAC GGT CAA AC-3′ and 5′-CCG AGT GTG GTT TGG AGA TT-3, as described previously 34 , were used to amplify each region of approximately 780 bp of the 28S gene and 790 bp of the per gene, respectively. Polymerase chain reaction (PCR) was performed to amplify these target regions in accordance with the procedure reported together with primers except using TaKaRa Ex Taq (Takara Bio Inc., Japan). PCR products were confirmed by gel electrophoresis and purified using a QIAEX II Gel Extraction Kit (QIAGEN). PCR products were then subjected to sequence analysis (Fasmac Co., Ltd., Japan) using the same primers. Sequence data was analyzed for similarity using NCBI Nucleotide BLAST. Feeding assay. For the feeding assay, first instar larvae hatched from the eggs were used. Each diet (16-40 g; pork-beef mince, pork fat mince, human fat, skin, muscle mince, and human necrotic tissue mince) was mixed with half distilled water and served to 30-100 first instar larvae. These larvae were kept at 27 °C in the cage and developed into adults. The number of surviving individuals (larvae, pupae, and adults) was counted to calculate the survival rate.
The weight of each individual and its developmental stage (larvae, pupae, and adults) were evaluated. The weight of each individual was measured using an electronic microbalance MSA6.6S-000-DM (Sartorius AG, Göttingen, Germany) with an accuracy of 0.001 mg. The dry weight of larva was measured following lyophilization of larvae using a freezing vacuum drying machine FreeZone 4.5 (Labconco Co., Kansas City, MO, USA).
The stage of each larva was determined using the morphological feature and behavior of the larvae. Images of the posterior spiracular slits of each larva were captured using a stereomicroscope equipped with a camera as described above. A larva carrying three separated spiracular slits and a complete peritreme with an inter slit projection was judged as a third instar larva (early and late). Of these larvae, a larva that stopped feeding and wandered on dry areas rather than wet ones was considered as a late stage of the third instar.
The amount of diet ingested by larvae was determined as follows. First, 6 g of diet was served to 10 first instar larvae, and the diet remaining at 5 days after hatching was measured using an electronic microbalance. Then, the net amount of the ingested diet was divided by the number of larvae to calculate the food consumption per larva.
Body size, wing vein length, and trichome density. Body size, vein length, and trichome density in the wings were compared among fly strains. Thirty first instar larvae were fed on 18 g of pork-beef mince. The pupae and adults were collected at 11 days and 26 days after hatching, respectively. Images of the pupal and adult body were captured using a stereomicroscope equipped with a camera as described above. Pupal and adult body lengths from the vertex of the head to the posterior tip of the abdomen were measured. Adult wings were washed three times with a solution containing 70% ethanol and 0.3% Triton X and mounted in 80% glycerol. Images of adult wings were captured using a Leica DM2500 microscope equipped with Leica DFC300 FX digital camera (Leica Microsystems GmbH, Wetzlar, Germany). The dm-cu length of the wing vein of female adults was measured. Trichome density in a 150-pixel square was calculated using the freeware Fijiwings (https:// sourc eforge. net/ proje cts/ fijiw ings/).

Immunohistochemistry.
The cell size of the larval fat body was examined as follows. Ten larvae at 4 days after hatching were collected on an iced petri dish and washed with ice-cold phosphate-buffered saline (PBS). The larvae were dissected in PBS and then fixed in 4% paraformaldehyde for 5 min on ice, then incubated for www.nature.com/scientificreports/ 60 min at room temperature. Fixed whole tissues were washed with PBT (PBS + 0.1% Triton X-100) three times for 20 min each and incubated with PBTn (PBT + 5% goat serum) for 30 min at room temperature. The tissue was washed with PBT 4 times for 10 min each and incubated with Alexa Fluor 555 Phalloidin (Thermo Fisher Scientific, MA, USA) containing 1% BSA for 2 h at room temperature. The tissue was washed with PBT 4 times for 10 min each and incubated with TO-PRO-3 iodide (Thermo Fisher Scientific, MA, USA) for 15 min. The tissue was washed with PBT 4 times for 10 min each and kept in PBS at 4 °C. The fat body was collected from the tissue and mounted using Vectashield (Vector Laboratories, CA, USA). Images were acquired on a confocal microscope TCS-SP8 (Leica Microsystems GmbH, Wetzlar, Germany). Ten fat body cells per individual larva were used to measure cell size using NIH Image-J (https:// imagej. nih. gov/ ij/).

Larval excretions and secretions (ES).
Fly eggs were washed with ultrapure water, 1% sodium hypochlorite solution, 70% ethanol, and ultrapure water. The washed eggs were placed in a plastic tube. One hundred first instar larvae that hatched from eggs were incubated in 200 μl of PBS for 1 h at room temperature. The supernatant containing larval ES was collected in a 1.5 ml tube. The larval ES solution was concentrated using a centrifugal filter Amicon Ultra-0.5 (UFC500324, Merck Millipore, Darmstadt, Germany Fibrin dissolution assay. Ten milliliters of 5 mg/ml fibrinogen solution dissolved in 0.2 M sodium borate buffer (pH 7.8) (067-03693, Wako Pure Chemical Co., Tokyo, Japan) were added to a petri dish (100 mm in diameter). Five hundred microliters of 20 unit/ml human thrombin solution in a 0.2 M sodium borate buffer (33839-46, Nacalai Tesque Inc., Kyoto, Japan) was added to the dish, stirred immediately, incubated for 30 min to form a coagulated fibrin plate. Fifty microliters of each cell culture supernatant collected in the scratch assay (as described below) were added to the center of the plate. The plates were incubated for 40 h at 37 °C. Images of the fibrin-dissolved area were captured, and the dissolved area was measured using NIH Image-J.
In vitro scratch assay. HFF cells (0.6 × 10 4 per well) in a 24-well plate were cultured at 37 °C. After forming a confluent cell monolayer, the cells were scraped with the top of a 200 μl pipette tip in a straight line to create an artificial gap (scratch). The debris from the scratched cells was removed, and the edge of the scratch was smoothed by washing the cells twice with PBS. Five hundred microliters of DMEM containing 1% FBS and 10 μg/ml gentamicin were added to each well. Fifty µl of PBS or larval ES solutions diluted to adjust the protein concentration with PBS were supplemented into the medium. After 12 h, the cell culture supernatants were collected and kept at − 80 °C for the fibrin dissolution assay (as described above). Images of each well after 0 or 12 h of culture were captured, and the closure areas (cell-covered gap) were measured by comparing the two images for each well using NIH Image-J. The closure areas of the PBS-treated cells at 12 h were adjusted to have a mean value of 100. The closure areas of samples were calculated by dividing each value by that of PBS-treated one obtained in the same experiment.
De novo assembly and analysis of RNA-seq data. Fly eggs were washed with ultrapure water, 1% sodium hypochlorite solution, 70% ethanol, and then ultrapure water again. Washed eggs were placed in a plastic tube. The first instar larvae that hatched from the eggs were collected for RNA-seq analysis. TRIzol (Invitrogen, CA, USA) was used to isolate the total RNA from these larvae. The total RNA samples were sequenced on an Illumina HiSeq 2500 (100 bp, paired-end) and analyzed by Hokkaido System Science Co. Ltd. (Hokkaido, Japan). Sequenced reads were trimmed using Cutadapt 35 and Trimmomatic 36 . Because any reference genome or transcriptome datasets of L. sericata were not available at the beginning of this study, de novo transcriptome assembly of RNA-seq data was performed using Trinity 37 . Gene expression levels among fly strains were estimated with RSEM 38 and compared using edgeR 39 . Assembled transcripts were manually annotated, referring to available genetic information of other brachyceran fly species.
Statistical analysis. Student's t-test and two-sided log-rank test were performed in this study using R version 3.4.1 (http:// www.r-proje ct. org). Probability values less than 0.05 were considered statistically significant.

Results
Development of a human tissue-based feeding assay for fly larvae. The feeding and growth characteristics of fly larvae when fed on human tissues were examined at first. Human tissues prepared as medical waste from surgery were sorted according to organ type, minced, and supplied as a diet for larval feeding (Fig. 1a). A different amount of human skin mince was supplied to fly larvae collected immediately after hatching (20 individuals each). To evaluate larval growth, larval weight was measured 4 days after hatching, when most of www.nature.com/scientificreports/ the flies became wandering third instar larvae. As a result, any human skin mince used in this experiment was able to support larval growth, increasing their weight (Fig. 1b). Larvae fed with 12-16 g of human skin mince did not show any additional increase in weight compared with larvae fed with 8 g (8 g vs. 12 g: p = 0.22, 12 g vs. 16 g: p = 0.69, Fig. 1b), suggesting that feeding with 0.4 g of human skin mince is enough for one larva to grow fully. Next, the effect of feeding human and pork tissues (fat mince) to fly larvae was compared on their growth and survivability. Pork-beef meat mince was used as a standard diet. The weight of larva fed with human fat was significantly higher than that of larva fed with pork fat (p < 0.01 at 4 days after hatching, Fig. 1c). The survival rate of larvae fed with human fat was equivalent to that of larva fed with pork fat (p = 0.45, Fig. 1d). The pork-beef mince showed higher values of both growth and survival than human or pork fat mince (Fig. 1c,d). These results suggested that the nutritional value of the human tissue is comparable to or higher than pork tissue.
Human tissue-dependent growth and survivability of fly larvae. To examine which human tissues are appropriate for feeding flies, larvae were fed with different minces prepared from human fat, skin, and muscle. Pork-beef meat mince was used as a standard diet. Larval weights at 4 days after hatching varied depending on which tissue the larvae ingested; human muscle mince supported larval growth to a similar level as the pork- www.nature.com/scientificreports/ beef meat mince, whereas human fat mince showed a relatively low ability to support larval growth (Fig. 2a). The survival ratio of larvae fed on human muscle mince was significantly higher than that for other human tissues (p < 0.01, Fig. 2b). It was also observed that the transition of larval developmental stages was significantly affected by the tissue that larvae ingested. Larvae fed with human muscle developed into third instar larva as early as ones fed on pork-beef meat mince (Fig. 2c,d). On the other hand, although most of the larvae fed on human skin or muscle already started wandering at 4 days after hatching, larvae fed on human fat showed a significant delay in development (p < 0.01, Fig. 2c). No wandering larvae (late third instar) raised on human fat were observed (Fig. 2d), suggesting that human fat itself is insufficient to support proper larval development. Next, the feasibility of a human tissue-based feeding assay was considered in terms of the availability of human tissues. The results indicated that human muscle was the most nutritious diet appropriate for larval growth and development, and the human skin was the next best ( Fig. 2a-d). Given the practical difficulty in securing human muscle tissues as medical waste, the human skin, frequently obtained from flap surgeries, was used as a diet resource for the human tissue-based feeding assay in this study.  (Table 1). Fly adults emerged from larvae on 23 corpses, and characterization of the morphological traits identified adults from 14 corpses as L. sericata (Fig. 3a). Four strains (#28, #34, #35, #36) were established from independent corpses and subsequently confirmed as L. sericata by analyzing nucleotide sequences of the mitochondrial COI, nuclear 28S rRNA, and period genes. Larvae of these corpse-bone strains were fed on human skin mince or pork-beef mince, followed by measuring the weight of each larva. Each larva of the four corpse-derived strains was 1.33-1.58 times heavier than the control strain after feeding on human skin mince (p < 0.01, Fig. 3b). A similar result was obtained from the experiment using the pork-beef mince (Fig. 3b). To avoid overestimating the water content in the larval body, the dry weight of each larva was measured. Significant differences were also observed among larvae of two corpsederived strains (#28 and #36) compared with the control strain (p < 0.01, Fig. 3c). These results suggested that L. sericata strains sourced from the human corpses may be comparable to or better than standard medical strains in the consumption of human tissues.
A corpse-derived fly strain that ingests human tissue effectively. One of the corpse-derived strains (strain #28), which gained significantly more weight than the control when fed on human skin (Fig. 3b,c), was selected and subjected to further analysis. Strain #28 grew faster and molted into the third instar larva earlier than the control (Fig. 4a,b), indicating that these larvae may ingest a larger amount of human tissue. To evaluate the larval feeding ability more accurately, the food intake was quantified by measuring the remaining diet after feeding. The amount of pork-beef mince or human skin mince consumed by larvae of strain #28 was significantly larger than the control at 5 days after hatching (p < 0.01, Fig. 4c), confirming that food intake per unit time differed according to the fly strain. Strain #28 significantly grew heavier than the control when fed on necrotic tissue (p < 0.01, Fig. 4d), indicating its substantial feeding ability on necrotic tissues. These results suggested that corpse-derived strain #28 has a higher feeding ability on human tissues and can be of practical use for MDT.

Correlation between feeding ability and body size of corpse-derived flies. Higher nutrition
intake from ingested human tissues may correlate with various fly phenotypes such as physical enlargement, increasing basal metabolic rate, or precocious development. To clarify if the higher feeding ability of the corpsederived strain related to other features besides faster development, body sizes among the strains were compared. Instead of larva, the body lengths of pupae and adults were measured because preservation may hinder accurate measurement of larval body length 40 . The body sizes of pupae and adults of strain #28 were significantly larger than the control, consistent with the results from measuring their weights (Fig. 5a,b).
Then, the cell size of strain #28 was analyzed to examine whether a difference in cell size or cell number contributed to the body size enlargement. Each epidermal cell of the fly wing has a single hair, called a trichome; therefore, the density of trichomes indicates cell size. The number of trichomes within a defined wing area of strain #28 was lower than that of the control (Fig. 5c). It was also observed that the length of a wing veins of strain #28 was longer than that of the control (Fig. 5c), suggesting the presence of larger cells in the body of strain #28. As expected, larger cells were also present in the fat body of strain #28 (Fig. 5d). In addition to strain #28, the other "big eater" strains also showed large adult body sizes (Fig. 5e). These results suggested that the body size of carrion flies can be a reliable marker for their potential for food consumption. www.nature.com/scientificreports/ Cell proliferation activity of larval excretions and secretions. It has been assumed that larval ES are essential to enhance wound healing by stimulating human cell proliferation and migration 16,18 . To evaluate the capability of strain #28 in wound repair, the proliferation and migration rate of HFF cells treated with its larval ES were measured. The larval ES-stimulated cellular activity of fibrinolysis (dissolution of fibrin clots) was also examined. Each larval ES collected from strain #28 and the control was confirmed to promote the proliferation of HFF cells and dissolved fibrin clots (Fig. 6a,b). The proliferation rates of HFF cells incubated with the larval ES of strain #28 were higher than those of the control at concentrations of 10, 25, and 50 μg/ml (Fig. 6a). On the other hand, the fibrin dissolution activity of HFF cells induced by the larval ES of strain #28 was similar to the control larval ES (Fig. 6b). It was noted that no effect on cell migration rate was observed when the larval ES of strain #28 or the control was added to HFF cells (Fig. 6c). Finally, to obtain molecular insight into the higher ability of strain #28, the de novo transcriptome assemblies of RNA-seq data in first instar larvae immediately after hatching were compared between strain #28 and the control. Thresholding genes with q values < 0.05 yielded 2,993 genes as differentially expressed genes (DEGs); www.nature.com/scientificreports/ 1,623 genes were upregulated, and 1,370 genes downregulated in strain #28 (Fig. 6d). A manual annotation predicted that the DEGs might contain heat-shock proteins, ribosomal proteins, glutathione S-transferases, cytochromes P450, cuticle associating factors, and insulin-like peptide signaling pathway associating factors. This result implied that the fly abilities such as debriding damaged tissue and enhancing granulation tissue formation may be achieved by the differentially regulated genes in each strain.

Discussion
MDT has long been used for the treatment of necrotic wounds without refining fly species and strains due to the lack of available methods to evaluate the fly's ability in debriding human tissues. In this study, adopting medical waste from surgery on human patients opened the way to measure how much larvae ingest human substrates for the first time. In addition to previous studies 41,42 , larval growth rates have been demonstrated to vary depending on the animal species and the type of tissues they intake, suggesting that applying human tissue instead of edible meat from livestock is needed for selecting fly strains toward improving MDT. In particular, a feeding assay using human skin, in which the elasticity and viscoelasticity are unique 43 , can be a highly representative model for MDT.
To the best of our knowledge, this is the first modern study employing human corpses as a resource for wild flies to establish new strains applicable for MDT. L. sericata is known for its beneficial uses in forensic entomology and is one of the primary insects from cadavers used as an indicator to estimate the postmortem interval 44 . www.nature.com/scientificreports/ Development time at a particular temperature varies among L. sericata populations depending on the local climate where the fly was obtained, suggesting the occurrence of adaptation of flies to their environment and possibly altering the fly's phenotype 45 . Indeed, our study revealed that the feeding amount and growth rate of larvae, which is essential to determine the application interval for MDT, varies among fly strains collected from human corpses. Notably, the control strain, which has been maintained for a long time in the laboratory environment, showed a relatively lower capability of debridement than any of the corpse-derived strains examined. Adaptation of the control strain to artificial breeding conditions through the mechanism of phenotypic plasticity may be considered 46,47 . It is also possible that fly larvae of the corpse-derived strains, which have ancestors that have recently experienced feeding on human tissues, may prefer necrotic human carrion resulting in ingesting a larger amount of these tissues. Variations in feeding amount and growth rate among the flies of even the same species suggested the importance of evaluating fly strains before and after their application for MDT. Larvae of corpse-derived L. sericata strain (#28) ingested 40% more human tissue, grew larger, and reached the third instar and pupal stage almost 1 day earlier than the conventional medical maggots. Only first and early second instar larvae are applied to wounds or ulcers in clinical practice, so larvae ingesting a larger amount of tissue at the early stage may be beneficial for MDT. The enlarged cell size observed in the larval fat body and adult wing of the corpse-derived strain may explain its increased appetite and accelerated growth, although it remains unclear whether the cell enlargement is a cause or a consequence of its higher food intake ability. In  www.nature.com/scientificreports/ MDT treatment, the maggots are usually replaced every 48 h and repeated for one to three courses, depending on the size of the wound surface. Application of the "gluttonous" maggots identified in this study to MDT may reduce the number of repeated courses, thus suggesting that the burden on the patient can be reduced. Another noteworthy feature observed in the corpse-derived strain was a remarkable effect of larval ES on the proliferation of HFF cells compared to the control strain. The effects of larval ES on the proliferation and migration of human fibroblasts have been previously proposed 16,18 . The larval ES may further promote granulation tissue formation by stimulating the proliferation of fibroblasts in human wounds. However, details of larval ES function, such as which compounds in larval ES exert wound-healing activity 48 , remain unknown and need further investigation. A set of candidate genes possibly related to its higher abilities of larval feeding and stimulating cell proliferation was identified by analyzing DEGs in the corpse-derived strain. Notably, a tribbles-like gene was annotated as being more highly expressed in the corpse-derived strain. Drosophila Tribbles was first identified as pseudokinase playing roles in regulating cell proliferation and migration 49 . Contrary to the previous findings that Drosophila Tribbles regulates insulin signaling negatively and its overexpression led to decreased larval size and delayed maturation 50 , L. sericata Tribbles may function to enhance cell growth signaling at least in the larval stages. Indeed, complicated roles of Tribbles, occasionally transforming from a tumor suppressor gene into an oncogene, have been reported in several species, including humans and mice 51 . The germ-line transformation method for L. sericata has been established; a transgenic fly expressing human platelet-derived growth factor-BB was reported 52,53 . Genome editing in L. cuprina, a close relative of L. sericata, using the CRISPR/Cas9 system has also been reported, and its efficacy was demonstrated by disrupting the yellow gene 54 . Gene manipulation targeting these genes in L. sericata may provide detailed information about the cues responsible for eliciting the ingestion of more tissue and faster growth. Moreover, it may enable the establishment of manipulated high-performance fly strains appropriate for improved MDT to treat wounds and ulcers more effectively.
In this study, we established novel carrion blowfly strains, and a detailed analysis of the maggots' feeding rate, wound healing effect, and gene expression suggested that they have the potential to be used for more effective MDT. All the results reported here are from in vitro experiments, and further in vivo analysis of therapeutic effects is expected for use in MDT on actual clinical applications.

Conclusion
The present study showed that a newly established feeding assay using human tissue may be preferred to evaluate important parameters in medical maggots, focusing mainly on the amount of food ingested by carrion fly larvae. It was also revealed that the corpse-derived strains had higher feeding ability, and one of these strains was capable of stimulating human cell proliferation via its larval ES more efficiently than the standard medical strain. Validating the feeding ability of medical maggots by employing human tissues and introducing new fly strains collected from human corpses may offer a promising strategy to secure and improve the quality of fly larvae, which may affect the outcome of MDT.